Medicago truncatula Mt-ZFP1 encoding a root enhanced zinc finger protein is regulated by cytokinin, abscisic acid and jasmonate, but not cold.

A Medicago truncatula zinc finger protein cDNA (Mt-ZFP1) was isolated from a M.truncatula seedling cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of Mt-ZFP1 is over 79% similar to S-SCOF-1 from soybean, a novel cold-inducible zinc finger protein involved in cold stress signal transduction mediated by abscisic acid (ABA). The secondary structure of Mt-ZFP1 protein was almost identical to that of S-SCOF-1. Mt-ZFP1 also contained a typical C2H2-type zinc finger domain and a putative nuclear located signal. RNA gel blot hybridization demonstrated that the Mt-ZFP1 gene was actively expressed in roots, with a lower abundance in leaf and stem tissues. Cold treatment did not induce the expression of Mt-ZFP1 in either leaves and stems or roots. Exogenous application of cytokinins marginally increased the accumulation of Mt-ZFP1 mRNA, while ABA and jasmonate treatments decreased the levels of Mt-ZFP1 mRNA. DNA gel-blot analysis demonstrated that Mt-ZFP1 is present as a single copy gene in the M. truncatula genome. These data suggest that Mt-ZFP1 is a novel zinc finger protein with different physiological functions to that of S-SCOF-1. The similar cold-inducible factor like S-SCOF-1 might not exist in M. truncatula.     

The Medicago truncatula small protein proteome and peptidome

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago truncatula: the root tip and root of germinating seedlings, nitrogen fixing nodules, and young leaves. The isolation and fractionation strategies successfully enriched the small protein and native peptide content of the samples. Eighty-one small M. truncatula proteins and native peptides were identified. Most samples were dominated by ribosomal and histone proteins, and leaf samples possessed photosynthesis-related proteins. Secreted proteins such as lipid transfer proteins were common to several tissues. Twenty-four hours after germination, the roots and root tip tissues possessed several "seed-specific" and late-embryogenesis proteins. We conclude that these proteins are present in cells prior to germination and that they are subsequently used as a nutritional source for the young tissues. Native UV absorbing peptides were detected in very low molecular weight fractions and sequenced. Each peptide shared C-terminal residues and showed homology to the seed storage protein legumin. The strategies used here would be suitable for combining bioassays and mass spectrometry to identify bioactive peptides in the M. truncatula peptidome.     

An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate.

We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase. DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases. DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone. RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings. LOX1 expression levels were highest in roots and young seedlings. In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h. Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae. LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid. Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h. When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.     

Identification and characterization of MtMTP1, a Zn transporter of CDF family,in the Medicago truncatula

Zn is an essential micronutrient in plants, and the mechanisms of Zn homeostasis are under intensive study. In this report, we have identified MtMTP1, a Zn transporter of the CDF family in the legume model plant Medicago truncatula. The ORF of the MtMTP1 cDNA encodes a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. Like other metal tolerance proteins (MTPs) in plants, heterologous expression of MtMTP1 can complement the Zn-susceptible zrc1 cot1 yeast double mutant. The expression pattern was studied by quantitative fluorescent PCR. The expression of MtMTP1 was detected in all vegetative organs with the highest level of expression observed in leaves. With Zn supplementation its expression in roots was reduced while its expression in stems was increased in the first 2 days. No obvious changes were detected in leaves. Inoculation with Rhizobium meliloti down-regulated its expression in roots.     

Isolation,sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase

The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.     

The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi

The MtSucS1 gene encodes a sucrose synthase (EC 2.4.1.13) in the model legume Medicago truncatula. To determine the expression pattern of this gene in different organs and in particular during root endosymbioses, we transformed M. truncatula with specific regions of MtSucS1 fused to the gusAint reporter gene. These fusions directed an induction to the vasculature of leaves, stems, and roots as well as to flowers, developing seeds, young pods, and germinating seedlings. In root nodules, strong promoter activity occurred in the infected cells of the nitrogen-fixing zone but was additionally observed in the meristematic region, the prefixing zone, and the inner cortex, including the vasculature. Concerning endomycorrhizal roots, the MtSucS1 promoter mediated strongest expression in cortical cells harboring arbuscules. Specifically in highly colonized root sections, GUS-staining was furthermore detected in the surrounding cortical cells, irrespective of a direct contact with fungal structures. In accordance with the presence of an orthologous PsSus1 gene, we observed a comparable regulation of MtSucS1 expression in the grain legume Pisum sativum in response to microbial symbionts. Unlike other members of the MtSucS gene family, the presence of rhizobial or Glomus microsymbionts significantly altered and enhanced MtSucS1 gene expression, leading us to propose that MtSucS1 is involved in generating sink-strength, not only in root nodules but also in mycorrhizal roots.     

Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh

A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.     

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.     

Characterisation and expression studies of a root cDNA encoding for ferredoxin-nitrite reductase from Lotus japonicus

A full-length cDNA encoding for ferredoxin-nitrite reductase (NiR, EC 1.7.7.1), has been isolated from a root cDNA library from the legume Lotus japonicus and characterised. The NiR gene ( Nii ) is present as a single copy in this plant, and encodes a protein of 582 amino acids. The Lotus NiR protein is synthesised as a precursor with an amino-terminal transit peptide consisting of 25 amino acid residues. Sequence comparisons with leaf NiRs from different plant species and with other related redox proteins identified in the root NiR the same highly conserved residues involved in the cofactor binding than previously reported for leaves. Besides, a putative binding site for ferredoxin was also found in the N-terminal region of the protein. The NiR gene is expressed in roots and leaves, although the level of expression is much higher in roots, in accordance with the fact that L. japonicus assimilates nitrate mainly in roots. NiR mRNA, protein and activity are induced by nitrate in roots and leaves, while ammonium-grown plants only showed basal levels. No oscillations of NiR mRNA, protein and activity were observed during the day/night cycle, neither in roots nor leaves, making an interesting difference with rhythms observed in other plant species.